A ligand binding assay is an analytical technique for measuring concentrations of substances that react selectively with specific binding proteins. Such substances are generally called ligands in the field of biochemistry. Immunoassays to measure concentrations of antigens that react selectively with specific antibodies comprise a class of ligand binding assays.
Assay reagents in a ligand binding assay generally include:
1. A specific binding protein such as an antibody (Aby) that specifically and strongly binds the substance to be measured, referred to as the analyte (A), the strong binding being characterized by an equilibrium dissociation constant less than about 10.sup.-8 M; and PA1 2. Labeled analyte (A.L).
The label (L) provides a measurable property of the system for the purpose of monitoring the extent of binding of A.L to Aby in a solution.
The basic assay principle is illustrated by the equilibrium relationship EQU A.L+A--Aby.revreaction.A.L+Aby+A.revreaction.A.L--Aby+A (1)
where the hyphen indicates binding. Thus, in accordance with Eq. (1), A and A.L compete for binding to Aby. The more A present in a solution containing A.L and Aby, the less A.L will be bound to Aby. An assay is conducted by adding a sample containing an unknown amount of A to a solution which contains a known concentration of A.L. A measured amount of Aby is then added. The amount of A in the sample may be deduced by measurement of the concentration of bound A.L.
Radioactivity is a known label employed in immunoassays. Use of radioactive labels, however, requires physical separation of bound and free labeled analyte by procedures involving protein precipitation or removal of free label with particulate adsorbents. The separation procedures are time-consuming and contribute significantly to assay imprecision.
Since the inception of radioimmunoassay about twenty years ago, other labels have come into use. Some of the more recent labels have the desirable characteristic of having a measurable property that is significantly different for bound and free labeled analytes so that physical separation is unnecessary. Assays not requiring separation are known as "homogeneous".
Fluorescent dyes have been shown to be useful as labels in homogeneous immunoassays as described in J. F. Burd, "Fluoroimmunoassays in Drug Monitoring", Clinical Chemistry News (April 1982), p. 33. The life-time of the excited state and the rate of rotary motion of a dye molecule is such that the polarization of free labeled analyte is low, and the polarization of labeled analyte bound to a large protein is high. Fluorescence polarization is accordingly known to be a useful response variable as described in M. E. Jolley, et. al., "Fluorescence Polarization Immunoassay I", Clinical Chemistry 27 (1981), p. 1190. Determination of polarization requires measurement of two intensities and the use of polarizers in the excitation and detection beams with consequent reduction in signal intensity by about a factor of 10. Polarization immunoassay also requires a specially designed fluorometer, which is expensive.
Measurement of intensity differences is simpler and potentially more sensitive than polarization measurements. Significant intensity differences between bound and free labeled analyte, however, do not generally occur. Only in the case of thyroxin, as the analyte, has the intensity of the bound label been found to be significantly different (3x) from that of the free label, as shown in D. S. Smith, "Enhancement Fluoroimmunoassay of Thyroxine," FEBS Letters 77 (1977) p. 25. An intensity difference of 20% between bound and free fluorescein labeled gentamicin has been reported in E. J. Shaw, et al., "Estimation of Serum Gentamicin by Quenching Fluorimmunoassay," Journal of Clinical Pathology 30 (1977) p. b 526, to serve as the basis of a homogeneous response variable. The Shaw, et al. measurements required subtraction of fluorescent background from control samples containing antibody. The magnitude of the background subtraction was comparable to the intensity difference measured, thereby limiting the precision with which gentamicin concentration could be determined.
Because of the potential advantages of intensity measurements, efforts have been directed toward developing methods that would cause the intensity of free and bound label to differ more markedly. One known method is based upon the phenomenon of excitation energy transfer and requires conjugation of a complementary acceptor dye to the antibody as described in E. F. Ullman, et al., "Fluorescent Excitation Transfer Immunoassay," Journal of Biological Chemistry 251 (1976) p. 4172. When two dyes, with properly matched spectral properties, are sufficiently proximate, the excitation energy of the donor dye is transferred to the acceptor dye. If the acceptor dye is nonfluorescent, then the energy transfer results in reduced fluorescence intensity. The distance across which the energy can be effectively transferred from one dye to the other is such that labeled analyte must be bound to the antibody which has been conjugated with the acceptor dye. Therefore, bound labeled analyte is preferentially quenched and free labeled analyte is fully fluorescent. A disadvantage of this method is the need to conjugate acceptor dye to the antibody. Also, the amount of acceptor dye conjugation must be large enough to assure proximity to the binding site which is located at one end of a relatively large, elongated protein. On the other hand the amount of conjugation must not be so large that the binding affinity of the chemically altered antibody is substantially reduced. As a result, the criticality of the acceptor dye conjugation makes the dye-antibody conjugate difficult to manufacture and makes the excitation energy method expensive.
Another known method involves the use of an additional antibody generated against the label, as described in R. F. Zuk, "Fluorescence Protection Immunoassay," Clinical Chemistry 25 (1979) p. 1554. When some dye labels are bound to their antibodies, they no longer fluoresce. Furthermore, some such labels can bind to their antibodies only when free. Thus the free label is preferentially quenched because the dye moieties of the labeled analyte bound to the analyte antibody cannot bind to the label antibody. A disadvantage of this system is the need for an additional antibody which is expensive.
Accordingly, there is need for a simple and inexpensive ligand binding assay method using a nonprotein solute for preferentially altering the relative intensities of bound and free labels. The assay method should preferably be substantially independent of the particular ligand to be assayed.